Among the most remarkable bioactive compounds of the skin secretions of frogs are the antimicrobial peptides, which protect the amphibians against infections. Skin secretions from some frogs also contain proteases that can cleave the antimicrobial peptides into smaller molecules which may still be active. Since antimicrobial peptides are usually synergistic, the repertoire of similar molecules formed by proteolysis means the advantage of an optimized protection against pathogens at a low methabolic cost. We have characterized five antimicrobial peptides from the skin secretion of the Brazilian frog Leptodactylus ocellatus and have been observing the occurrence of shorter peptides derived from these five. Peptide breakage due to the methodology is an unlikely possibility, since secretions of other frog species have been treated by the same methods without revealing peptide fragments. Thus, we believe our finding actually bespeaks the occurrence of proteolytic activity in the skin secretion of L. ocellatus.
        Adult specimens of L. ocellatus collected in Distrito Federal region were kept at the University of Brasília. Skin secretion was obtained by mild electrical stimulation and lyophilized. Crude secretion aliquots (10.0 mg) were dissolved in 0.1% (v/v) TFA/water and fractionated by RP-HPLC using a C₁₈ column (Vydac 218TP1010). Elution was performed in a linear gradient of acetonitrile, at a flow rate of 2.5 mL/min. Chromatographic fractions were submitted to mass spectrometry analysis (MALDI-TOF) and some of them were further analyzed by automated sequencing (Edman degradation).
        We had already determined the N-terminal sequences of five antimicrobial peptides from the skin secretion of L. ocellatus, which ranged from 17 to 25 amino acid residues long and from 1730 u to 2559 u of molecular mass. We obtained the complete sequences of four smaller peptides derived from the former five. Two of these four have shown antibacterial activity against Staphylococcus aureus, in liquid broth assays. Other fifteen fragments have been detected by mass spectrometry (MALDI-TOF) analysis. Their molecular masses range from 970 u to 2034 u. Cleavages occur between positions 12 and 19 of the amino acid sequences, preferentially before or after a lysine residue. In order to confirm the presence of one or more proteases in the skin secretion of L. ocellatus, an electrophoretic assay will be performed to verify whether caseine is cleaved upon the presence of the crude secretion of Leptodactylus ocellatus. Should any protease be detected by electrophoresis, it will be submitted to peptide mass fingerprinting analysis.

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